Nuran Ogulener

Comparative study of the quercetin, ascorbic acid, glutathione and superoxide dismutase for nitric oxide protecting effects in mouse gastric fundus

The aim of this work was to compare the preventing capacity of quercetin with Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbic acid and glutathione on nitric oxide (NO)-induced relaxation in mouse gastric fundus. Furthermore, the effects of the quercetin on the tissue level of total oxidant and antioxidant was investigated. Nitrergic stimulation (4Hz, 25V, 0.1 ms, 10s-train) and exogenous NO (10 μM) induced relaxation. Pyrogallol (10 μM), hydroquinone (100 μM) and LY83583 (6-Anilino-quinolin-5,8-quinone, 5 μM) inhibited nitrergic relaxations. The inhibition observed with pyrogallol, hydroquinone and LY83583 was prevented by quercetin (0.1 μM). Also, ascorbic acid (500 μM), glutathione (100 μM) and Cu/Zn SOD (100 U/ml) prevented the inhibitory effect of superoxide anion generators on the relaxation to nitrergic stimulation and NO. Diethyldithiocarbamic acid (DETCA; 8mM) inhibited nitrergic relaxations. DETCA-induced inhibition on nitrergic stimulation and NO-induced relaxation was prevented by quercetin, ascorbic acid, glutathione or Cu/Zn SOD. DETCA plus pyrogallol, hydroquinone or LY83583 strengthened the inhibition on the relaxations. Also, pre-treatment with quercetin, ascorbic acid and glutathione prevented the inhibitory effect of DETCA plus LY-83583 on the relaxation to nitrergic stimulation and NO but Cu/Zn SOD did not prevent this inhibition. Also, quercetin increased tissue total antioxidant capacity and decreased tissue oxidant level and oxidative stress index in DETCA-treatment group. These results indicate that quercetin has antioxidant effect and protects NO from endogenous superoxide anion-driven inactivation and enhances its biological activity, suggesting that quercetin may scavenge superoxide anion in a Cu/Zn SOD, glutathione or ascorbic acid-inhibitable manner.

A putative role for S-nitrosoglutathione as the source of nitric oxide in photorelaxation of the mouse gastric fundus.

Mouse gastric fundus strips were relaxed by ultraviolet light (UV) irradiation, exogenous nitric oxide (NO), isoproterenol, S-nitrosoglutathione, S-nitroso-L-cysteine and S-nitroso-N-acetyl-penicillamine. Glutathione did not affect relaxations in response to UV irradiation, exogenous NO and isoproterenol while inhibiting that with S-nitrosoglutathione. L-Cysteine inhibited responses to UV irradiation and exogenous NO, but not in the presence of exogenous Cu(2+)/Zn(2+) superoxide dismutase. However, L-cysteine alone or in combination with Cu(2+)/Zn(2+) superoxide dismutase did not affect the relaxations in response to S-nitroso-L-cysteine. Ethacrynic acid and diamide inhibited photorelaxations but not the relaxations with exogenous NO and isoproterenol. This inhibition was prevented by glutathione, but not by L-cysteine. S-nitrosoglutathione-induced relaxations were abolished by diamide and ethacrynic acid, whereas responses to S-nitroso-L-cysteine and S-nitroso-N-acetyl-penicillamine were only inhibited by ethacrynic acid. These results suggest that S-nitrosoglutathione may, at least in part, be the putative S-nitrosothiol, which is converted to NO in response to UV irradiation in mouse gastric fundus strips.

Protective effect of quercetin, a polyphenolic compound, on mouse corpus cavernosum.

Flavonoids are plant‐based phenolic compounds, and quercetin is the most abundant dietary member of this family. One of the most important characteristics of quercetin is its antioxidant property. The aim of this study was to investigate antioxidant effects of quercetin on corpora cavernosa of mice. Corpora cavernosa were isolated in organ baths, precontracted with phenylephrine (0.5 μm) and relaxant responses were mediated by acetylcholine (0.1–1 μm), electrical field stimulation (EFS, 1–16 Hz, 0.5 ms, 30 V) or acidified sodium nitrite (a NaNO2, 0.5 mm). Superoxide anion generators; pyrogallol (50 μm), hydroquinone (100 μm), LY 83583 (6‐Anilinoquinolin‐5,8‐quinone, 10 μm) and superoxide dismutase (SOD) inhibitor; diethyldithiocarbamic acid (DETCA, 8 mm) were used in order to expose corpus cavernosa to oxidant stress. Acetylcholine (0.1–1 μm) induced relaxant responses were significantly inhibited in LY 83583 (10 μm) and DETCA + LY 83583 applicated trials. EFS‐induced relaxant responses were significantly inhibited in DETCA (8 mm) and DETCA + LY 83583 administrated trials. On the other hand, acidified sodium nitrite‐induced responses were inhibited by all of the superoxide anion generators tested. Quercetin (10 μm) failed to improve the inhibitions on endothelium and electrically stimulated responses. Acidified sodium nitrite (0.5 mm) mediated relaxant responses were significantly restored by quercetin except the groups in which LY 83583 were used. The data suggest that quercetin acts as a protective agent in mouse corpus cavernosum, increasing the bioavailability of exogenous nitric oxide by protecting it from superoxide anion (O2–).

Characterization of relaxant mechanism of H2 S in mouse corpus cavernosum.

The aim of this study was to investigate the mechanism of H2S‐induced relaxation in mouse corpus cavernosal tissue. l‐cysteine (10−6 × 10−3 mol/L) and exogenous H2S (NaHS; 10−6 to 10−3 mol/L) induced concentration‐dependent relaxation. l‐cysteine‐induced relaxations was reduced by d,l‐propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l‐cysteine induced relaxations, but not of those of H2S diminished in endothelium‐denuded tissues. Nω‐nitro‐l‐arginine (l‐NA; 10−4 mol/L), a nitric oxide synthase inhibitor, and ODQ (10−4 mol/L), a guanylyl cyclase inhibitor, increased the H2S‐induced relaxation. Zaprinast (5 × 10−6 mol/L) and sildenafil (10−6 mol/L), phosphodiesterase inhibitors, inhibited H2S‐induced relaxation. Adenylyl cyclase inhibitors N‐ethylmaleimide (2.5 × 10−5 mol/L) and SQ22536 (10−4 mol/L) reduced relaxation to H2S. Also, H2S‐induced relaxation was reduced by KCl (50 mmol/L), 4‐aminopyridine (10−3 mol/L), a Kv inhibitor, glibenclamide (10−5 mol/L), a KATP inhibitor or barium chloride (10−5 mol/L), a KIR inhibitor. However, H2S‐induced relaxation was not influenced by apamin (10−6 mol/L), a SKCa2+ inhibitor, charybdotoxin (10−7 mol/L), an IKCa2+ and BKCa2+ inhibitor or combination of apamin and charybdotoxin. Nifedipine (10−6 mol/L), an L‐type calcium channel blocker and atropine (10−6 mol/L), a muscarinic receptor blocker, inhibited H2S‐induced relaxation. However, H2S‐induced relaxation was not influenced by ouabain (10−4 mol/L), a Na+/K+‐ATPase inhibitor. This study suggests that H2S endogenously synthesizes from l‐cysteine by CSE endothelium‐dependent in mouse corpus cavernosum tissue, and exogenous H2S may cause endothelium‐independent relaxations via activation of K channels (KATP channel, KV channels, KIR channels), L‐type voltage‐gated Ca2+ channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H2S and NO/cGMP.

Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

AbstractAim:The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relaxation against superoxide anion inhibition in the mouse duodenum.Methods:The effects of exogenous SOD, N,N′-bis(salicylidene) ethylenediamine chloride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues.Results:Diethyldithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electrical field stimulation (EFS). SOD and ascorbate partially prevented the inhibitory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by EUK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exogenous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8.Conclusion:These results suggest that the nitrergic neurotransmission in the mouse duodenum is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endogenous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.

Selective modifiers of glutathione prevent restoration of photorelaxations in mouse gastric fundus

S‐nitrosoglutathione (GSNO) has previously been shown to have a role in ultraviolet (UV) light‐elicited relaxations and proposed to account for the photosensitive store in the mouse gastric fundus. Furthermore, the depletion of this photosensitive store and its replenishment via long‐term electrical field stimulation were demonstrated in the same tissue. In relation to these results, the aim of the present study was to investigate the putative role of S‐nitrosothiols in the restorative effect of long‐term electrical field stimulation on the reduced photosensitive store. Two series of UV light‐elicited relaxations (photorelaxations) were obtained, and the magnitudes of the responses were 53 ± 6 and 26 ± 3%, respectively. The second series of photorelaxations attenuated statistically when compared with those in the first series. Ethacrynic acid (1 μm), diamide (1 μm) and glutathione (1 μm) had no effect on the photorelaxations occurred in the second series of responses. Electrical field stimulation (4 Hz, 25 V, 1 ms, 60 min), applied between two series of photorelaxations, revealed a complete recovery of the attenuated photorelaxations appeared in the second series. NG‐monomethyl‐l‐arginine (100 μm), ethacrynic acid (1 μm) and diamide (1 μm) extensively prevented the restorative effect of electrical field stimulation on photorelaxations. In addition, glutathione (1 μm) reversed the prevention achieved by ethacrynic acid and diamide. The conclusion is that the restoration accomplished by electrical field stimulation is because of the activation of nitric oxide synthase, which in turn brings about the regeneration of GSNO proposed to be the photodegradable material store.

The effects of thiol modulators on nitrergic nerve- and S-nitrosothiols-induced relaxation in duodenum

Abstract Background: The aim of this study was to investigate whether thiols are involved in the nitrergic neurotransmission in mouse duodenum. Methods: The effects of thiol-modulating agents, ethacrynic acid (100 μM), a non-specific sulfhydryl alkylator, and diamide (100 μM), an alkylating agent that oxidizes protein sulfhydryl groups and depletes intracellular glutathione, on relaxations to nitrergic stimulation (electrical field stimulation, EFS;10 Hz, 25 V, 1 ms, 15 s-train), S-nitrosoglutathione (GSNO; 5 μM), S-nitroso-acetylpenicillamine (SNAP; 5 μM), and S-nitrosocysteine (CysNO; 10 μM) were investigated. Moreover, the effects of buthionine sulfoximine (100 μM), an inhibitor of γ-glutamylcysteine synthetase, and sulfobromophthalein (100 μM), an inhibitor of glutathione-S-transferase, were studied on relaxant responses to EFS and S-nitrosothiols in mouse duodenum. Results: Exogenous free thiol, glutathione (GSH, 100 μM) did not influence relaxation to EFS, GSNO, SNAP, and CysNO. Ethacrynic acid and diamide significantly decreased relaxation of duodenum to EFS, GSNO, SNAP, and CysNO. This inhibition was prevented by addition of GSH. Buthionine sulfoximine and sulfobromophthalein significantly decreased relaxation to EFS and GSNO but did not influence relaxation to SNAP and CysNO. The inhibitory effect of buthionine sulfoximine and sulfobromophthalein on the relaxant response to EFS and GSNO was prevented by addition of GSH. Conclusions: These results suggest that relaxation to nitrergic stimulation is thiol-dependent, and nitrosothiols, possibly S-nitrosoglutathione may play a role, as an intermediate compound in nitrergic neurotransmission in mouse duodenum.

Effects of ethanol on RhoA/Rho-kinase-mediated calcium sensitization in mouse lung parenchymal tissue.

Calcium sensitization by the RhoA/Rho-kinase (ROCK) pathway contributes to the contraction in smooth muscle. Contractile stimuli can sensitize myosin to Ca(2+) by activating RhoA/Rho-kinase that inhibits myosin light chain phosphatase activity. The present study was aimed at investigating the possible involvement of RhoA/Rho-kinase pathway in contractile responses to agonist (phenylephrine) and depolarizing (KCl) of mouse lung parenchymal tissues. Also, we investigated the effect of ethanol on RhoA/Rho-kinase pathway. Phenylephrine (10(-8)-10(-4) M) and KCl (10-80 mM) induced sustained contractions in parenchymal strips. Ethanol significantly attenuated the contractions to phenylephrine and KCl. The Rho-kinase inhibitors fasudil (5×10(-5) M) and Y-27632 (5×10(-5) M) inhibited contractions to in both control and ethanol-treated parenchymal strips. In addition, the relaxations induced by fasudil (10(-4) M) and Y-27632 (5×10(-4) M) on parenchymal strips contracted by phenylephrine but not KCl was decreased in ethanol-treatment group. Also, RhoA, ROCK1 and ROCK2 expressions were detected in mouse lung parenchymal tissue. In ethanol-treated group, expression of RhoA and ROCK1 but not ROCK2 decreased compared to control. Furthermore, ethanol causes apoptotic changes in alveolar type I epithelial cells of parenchymal tissue. These results suggest that RhoA/Rho-kinase signaling pathway plays an important role in phenylephrine- and KCl-induced Ca(2)(+) sensitization in mouse lung parenchymal tissue. Also, ethanol may be decrease phenylephrine- and KCl-induced contraction due to lowering the RhoA/Rho-kinase-mediated Ca(2+)-sensitizing by inhibiting RhoA/Rho-kinase pathway in parenchymal tissue. These results may be lead to important insights into the mechanisms of lung diseases due to alcohol consumption.

The role of arachidonic acid/cyclooxygenase cascade, phosphodiesterase IV and Rho-kinase in H2S-induced relaxation in the mouse corpus cavernosum

BACKGROUND Penile corpus cavernosum is an extremely vascularized tissue and cavernosal smooth muscle tone is regulated by the balance between contractile and relaxant factor. We investigated the possible role of arachidonic acid/cyclooxygenase cascade, phosphodiesterase IV (PDEIV) and Rho-kinase in exogenous hydrogen sulfide (H2S)-induced relaxation in mouse corpus cavernosum. METHODS The relaxant response to H2S (NaHS as exogenous H2S; 1-1000μM) were obtained in isolated mouse corpus cavernosum tissues which pre-contracted by phenylephrine (5μM). The effects of 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA; 10μM), a selective phospholipase A2 (PLA2) inhibitor, indomethacin (1μM), a non-selective cyclooxygenase (COX) inhibitor, baicalein (10μM), a lipoxygenase (LOX) inhibitor, and proadifen (10μM), cytochrome P450 inhibitor, on the relaxant responses to H2S were investigated. Furthermore, the effects of theophylline (500μM) and rolipram (1μM), a non-selective and selective PDEIV inhibitor, and fasudil (3μM), a specific Rho-kinase inhibitor, were studied on H2S-induced relaxation. RESULTS H2S-induced relaxations were significantly reduced by OBAA, indomethacin and proadifen but not baicalein. Furthermore, theophylline, rolipram and fasudil reduced H2S-induced relaxations. CONCLUSION These results suggest that PLA2, COX, cytochrome P450, PDEIV and Rho-kinase pathway may involve in H2S-induced relaxation in mouse corpus cavernosum tissues.